Endocytosis is required for many cellular pivotal processes including membrane recycling, nutrient uptake and signal transduction. This process is particularly relevant in polarized cells such as neurons. Exposure to ethanol causes a variety of anomalies in the developing brain and previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin (BSA) and transferrin (Tf) in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. The present study shows that the exposure of neurons in primary culture to chronic ethanol (0 to 14 DIV, 30 mM) affects both clathrindependent and clathrinindependent endocytosis by probably acting not only on the several proteins which are directly involved in the different steps of these processes, but also on the proteins that participate in the organization and dynamics of the actin cytoskeleton as well as on cholesterol cellular levels. Therefore, a toxic effect of alcohol on endocytosis could affect some of the important neuronal activities which depend on the endocytic process, including synaptic vesicle recycling, trafficking of postsynaptic receptors, polarized axon elongation, growth cone navigation and neuronal migration.